Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutation to the large and complex gene, PKHD1. Clinical and molecular studies have revealed a wide phenotypic range associated with PKHD1 mutation, from perinatal death to adult onset liver disease. Further complexity is provided by marked allelic heterogeneity and so only preliminary genotype/phenotype correlations have been possible. The first aim of this proposal will extend these mutation studies (and associations with phenotype) to further molecularly characterize typical and atypical ARPKD populations, including isolated liver disease. The role of genotype in dictating phenotype will be examined, and important residues highlighted for testing in functional studies. The ARPKD protein is large and membrane bound, and in common with other PKD associated proteins, localized to primary cilia and the basal body. Pkhdl defective animals have shortened and abnormal cilia. A fibrocystin paralog is found in humans, fibrocystin-like (-L;encoded by PKHDL1), and although there are hints at possible function, the precise role of this protein is unclear (it is not known to be associated with ARPKD). Fibrocystin-L is the most ancient member of the protein family and, unlike fibrocystin, present in fish and Chlamydomonas, where silencing results in impaired motility due to defective flagella;suggesting a ciliary role in other organisms. The second aim will use monoclonal antibodies and expression constructs to determine (and confirm) the subcellular localization of both proteins, and see if fibrocystin-L is associated with the ciliary/basal body axis. The cellular phenotypes associated with silencing these genes by shRNA, singly or in combination, in polarized kidney epithelial cells, will also be tested. In the third aim, in situ hybridization and morpholino approaches will be used to localize the sites of expression and the phenotype associated with knocking down activity in the developing zebrafish. Cystic phenotypes in the pronephric duct are one possible outcome, as is defective convergent extension movements, as found in zebrafish morphants of some syndromic PKD associated genes. The final aim will knockout PkhdH in the mouse, constitutively (and, if necessary, conditionally in the kidney) to determine the role of fibrocystin-L in mammals. Breeding with the existing Pkhdl deZ mouse will show whether loss of both fibrocystins results in an additive phenotype, suggesting related roles. Overall the project will reveal functional information about the roles of the fibrocystin protein family and identify signaling/developmental pathways involving these molecules.